LOCUS       sequence-260224-        5937 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Cre-dependent short hairpin RNA (shRNA) that targets the 
            3'-untranslated region of the rat Nts gene which encodes 
            neurotensin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5937)
  AUTHORS   Pomrenze MB, Giovanetti SM, Maiya R, Gordon AG, Kreeger LJ, Messing 
            RO
  TITLE     Dissecting the Roles of GABA and Neuropeptides from Rat Central 
            Amygdala CRF Neurons in Anxiety and Fear Learning.
  JOURNAL   Cell Rep. 2019 Oct 1;29(1):13-21.e4. doi: 
            10.1016/j.celrep.2019.08.083.
  PUBMED    31577943
REFERENCE   2  (bases 1 to 5937)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2019.08"; journalName: "Cell Rep"; date: 
            "2019-10-1- 1"; volume: "29"; issue: "1"; pages: "13-21.e4"
COMMENT     Sequence Label: AAV8-hSyn-flex-miR30-eGFP-shNts
FEATURES             Location/Qualifiers
     source          1..5937
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        71..518
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(556..589)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(640..673)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     ncRNA           complement(710..836)
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(864..878)
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(906..1027)
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     CDS             complement(join(1076..1789,1790..1792,1793..1795))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1079..1789,1790..1792,1793..1795))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1121..1142)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1470..1489
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1729..1750
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1792..1801
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1818..1851
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    1902..1935
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1960..2548
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2013..2033)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2431..2442)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2580..3056
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2779..2798)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3096..3236
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3096..3225
                     /label=AAV2 ITR
     rep_origin      3311..3766
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3398..3417)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3608..3629
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3783..3802)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3902..3924
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3962..3980)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4048..4152
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4153..4221,4222..5013)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4371..4390)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5184..5772
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5673..5692
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(5834..5937,1..26)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ggccaactcc atcactaggg gttcctgcgg ccgcacgcgt gtgtctagac tgcagagggc
       61 cctgcgtatg agtgcaagtg ggttttagga ccaggatgag gcggggtggg ggtgcctacc
      121 tgacgaccga ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc
      181 ccctatcaga gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt
      241 cagcaccgcg gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca
      301 ctgaaggcgc gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg
      361 gtcgcgtccg cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg
      421 ggcacgggcg cgaccatctg cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg
      481 tgggcagcgg aggagtcgtg tcgtgcctga gagcgcagtc gagaaggtac cggatcctct
      541 agagtcgact ccggaataac ttcgtatagg atactttata cgaagttatg cagaatggta
      601 gctggattgt agctgctatt agcaatatga aacctcttaa taacttcgta tagcatacat
      661 tatacgaagt tatggcgcgc ctttaaacgc attagtcttc caattgaaaa aagtgattta
      721 atttatacca ttttaattca gctttgtaaa aatgtatcaa agagatagca aggtattcag
      781 ttttagtaaa caagataatt gctcctaaag tagccccttg aattccgagg cagtaggcaa
      841 catgtgattc tcatccttta ctacatctgt ggcttcacta gtaaaggatg agaatcacat
      901 ggcgctcact gtcaacagca atataccttc tcgagccttc tgttgggtta acctgaagaa
      961 gtaatcccag caagtgtttc caagatgtgc aggcaacgat tctgtaaagt actgaagcct
     1021 cattcaacat agtatatgtg ctgccgaagc gagcacttaa caaggcttgc ggccgctact
     1081 tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc agcaggacca
     1141 tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctgggtgctc aggtagtggt
     1201 tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag tggtcggcga
     1261 gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg atgccgttct
     1321 tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc agcttgtgcc
     1381 ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg ttcaccaggg
     1441 tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc ttgaagaaga
     1501 tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg tgctgcttca
     1561 tgtggtcggg gtagcggctg aagcactgca cgccgtaggt cagggtggtc acgagggtgg
     1621 gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc ttgccgtagg
     1681 tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca
     1741 gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc accatggtgg
     1801 cgaccggtag cgctagcata acttcgtata aagtatccta tacgaagtta tttgccttaa
     1861 cccagaaatt atcactgtta ttctttagaa tggtgcaaag aataacttcg tataatgtat
     1921 gctatacgaa gttatgaatt cgatatcaag cttatcgata atcaacctct ggattacaaa
     1981 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     2041 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     2101 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     2161 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     2221 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     2281 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     2341 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctatgttgc cacctggatt
     2401 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     2461 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     2521 cggatctccc tttgggccgc ctccccgcat cgataccgag cgctgctcga gagatctacg
     2581 ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag
     2641 tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct
     2701 tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt gggaagacaa
     2761 cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca caatcttggc
     2821 tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt
     2881 tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg
     2941 gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc tacccacctt
     3001 ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga
     3061 ttttgtaggt aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc
     3121 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     3181 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     3241 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     3301 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3361 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
     3421 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3481 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     3541 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3601 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
     3661 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
     3721 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     3781 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     3841 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3901 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     3961 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     4021 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4081 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4141 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4201 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4261 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     4321 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     4381 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     4441 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     4501 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     4561 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     4621 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     4681 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     4741 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     4801 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     4861 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     4921 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     4981 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     5041 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5101 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5161 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5221 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5281 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     5341 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5401 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5461 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     5521 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5581 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5641 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5701 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5761 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5821 tttgctcaca tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg
     5881 tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagt
//
